Keita Okayama

Hepatocyte Growth Factor Reduces Cardiac Fibrosis by Inhibiting Endothelial-Mesenchymal Transition

The purpose of this study was to investigate the effect of hepatocyte growth factor (HGF) on the pathogenesis of cardiac fibrosis induced by pressure overload in mice. Although cardiac fibrosis is attributed to excess pathological deposition of extracellular matrix components, the mechanism remains unclear. Recent reports revealed that &agr;-smooth muscle actin–expressing myofibroblasts are primarily responsible for fibrosis. It is believed that myofibroblasts are differentiated from resident fibroblasts, whereas the transformation of vascular endothelial cells into myofibroblasts, known as endothelial-mesenchymal transition, has been suggested to be intimately associated with perivascular fibrosis. Thus, we hypothesized that HGF prevents cardiac fibrosis by blocking these pathways. We analyzed the pressure-overloaded HGF-transgenic mouse model made by transverse aortic constriction. Human coronary artery endothelial cells and human cardiac fibroblasts were examined in vitro after being treated with transforming growth factor-&bgr;1 or angiotensin II with or without HGF. The amount of cardiac fibrosis significantly decreased in pressure-overloaded HGF-transgenic mice compared with pressure-overloaded nontransgenic controls, particularly in the perivascular region. This was accompanied by a reduction in the expression levels of fibrosis-related genes and by significant preservation of echocardiographic measurements of cardiac function in the HGF-transgenic mice (P<0.05). The survival rate 2 months after transverse aortic constriction was higher by 45% (P<0.05). HGF inhibited the differentiation of human coronary artery endothelial cells into myofibroblasts induced by transforming growth factor-&bgr;1 and the phenotypic conversion of human cardiac fibroblasts into myofibroblasts. We conclude that HGF reduced cardiac fibrosis by inhibiting endothelial-mesenchymal transition and the transformation of fibroblasts into myofibroblasts.

Hepatocyte growth factor attenuates renal fibrosis through TGF-β1 suppression by apoptosis of myofibroblasts.

Objective The progression of chronic kidney disease (CKD) is characterized by the persistent accumulation of extracellular matrix. Especially, α-SMA-positive myofibroblasts producing large amounts of TGF-β1 are considered to play a key role in interstitial fibrosis. Although hepatocyte growth factor (HGF) improved renal fibrosis in various models, the molecular mechanisms involved are not yet fully understood. Methods and results In this study, the molecular mechanisms of the inhibition of fibrosis by HGF was examined using HGF transgenic mice (HGF-Tg) with angiotensin II (Ang II) infusion in 4 weeks models. HGF-Tg mice showed significantly decreased Ang II-induced renal fibrosis and lesser numbers of interstitial myofibroblasts, whereas the antifibrotic effect of HGF was abrogated using HGF-neutralizing antibody. The antifibrotic action in HGF-Tg mice was concordant with a decrease in TGF- β1, collagen type I and IV mRNA expression and an increase in MMP-2 and MMP-9 expression. Furthermore, HGF-Tg mice treated with Ang II showed apoptosis of myofibroblasts. To further investigate the antifibrotic effect of HGF, cultured human mesangial cells were used. HGF induced apoptosis of myofibroblast. Inhibition of the FAK-ERK-MMP signaling cascade by specific inhibitor or siRNA significantly decreased HGF-induced myofibroblast apoptosis. Conclusion The present study demonstrates that the increase in metalloproteinases through FAK-ERK signaling by HGF promotes myofibroblast apoptosis. Activation of metalloproteinases by HGF in the fibrotic kidney might be considered to attenuate the progression of CKD.

Telmisartan Exerts Renoprotective Actions via Peroxisome Proliferator-Activated Receptor-γ/Hepatocyte Growth Factor Pathway Independent of Angiotensin II Type 1 Receptor Blockade

Angiotensin (Ang) II type 1 receptor blockers have demonstrated beneficial effects beyond blood pressure control in the treatment of chronic kidney disease. There is clinical evidence that telmisartan is more effective than losartan in reducing proteinuria in hypertensive patients with diabetic nephropathy, because it is a partial agonist of peroxisome-proliferator activated receptor-&ggr; (PPAR&ggr;), as well as an Ang II type 1 receptor blocker (AMADEO Study [A comparison of telMisartan versus losArtan in hypertensive type 2 DiabEtic patients with Overt nephropathy]). In this study, we examined the role of PPAR&ggr; activation in the renal protective actions of telmisartan using Ang II type 1 receptor–deficient mice. Renal injury was induced in Ang II type 1 receptor–deficient mice by producing unilateral ureteral obstruction, which exhibited severe renal interstitial fibrosis and inflammation. In these mice, telmisartan prevented hydronephrosis induced by unilateral ureteral obstruction more strongly than did losartan. Importantly, the prevention of renal atrophy and fibrosis by telmisartan was significantly attenuated by GW9662, a PPAR&ggr; antagonist. Interestingly, the downstream effector of PPAR&ggr; activation by telmisartan is hepatocyte growth factor (HGF), a well-known antifibrotic factor, because renal HGF expression was significantly increased by telmisartan, and a neutralizing antibody against HGF diminished the renal protective action of telmisartan. These beneficial changes by telmisartan were associated with a decrease in the expression of transforming growth factor-&bgr;1 and other proinflammatory and profibrotic cytokine genes through PPAR&ggr;/HGF activation. Our findings provide evidence of organ protective actions of telmisartan through the PPAR&ggr;/HGF pathway, independent of Ang II type 1 receptor blockade. Further development of the next generation of Ang II type 1 receptor blockers with added organ protective actions, such as PPAR&ggr; activation, might provide new beneficial drugs to treat renal and cardiovascular diseases.

Negative action of hepatocyte growth factor/c-met system on angiotensin ii signaling via ligand-dependent epithelial growth factor receptor degradation mechanism in vascular smooth muscle cells

Rationale: Neointimal hyperplasia contributes to atherosclerosis and restenosis after percutaneous coronary intervention. Vascular injury in each of these conditions results in the release of mitogenic growth factors and hormones that contribute to pathological vascular smooth muscle cell growth and inflammation. Hepatocyte growth factor (HGF) is known as an antiinflammatory growth factor, although it is downregulated in injured tissue. However, the precise mechanism how HGF reduces inflammation is unclear. Objective: To elucidate the mechanism how HGF and its receptor c-Met reduces angiotensin II (Ang II)–induced inflammation. Methods and Results: HGF reduced Ang II–induced vascular smooth muscle cell growth and inflammation by controlling translocation of SHIP2 (Src homology domain 2–containing inositol 5′-phosphatase 2), which led to Ang II–dependent degradation of epithelial growth factor receptor. Moreover, the present study also revealed a preventive effect of HGF on atherosclerotic change in an Ang II infusion and cuff HGF transgenic mouse model. Conclusions: These data suggest that the HGF/c-Met system might regulate extrinsic factor signaling that maintains the homeostasis of organs.

Hepatocyte Growth Factor Attenuates Transforming Growth Factor-β-Angiotensin II Crosstalk Through Inhibition of the PTEN/Akt Pathway

Both angiotensin II (Ang II) and transforming growth factor (TGF)-&bgr;1 are thought to be involved in the progression of chronic kidney disease. In contrast, hepatocyte growth factor (HGF) counteracts the actions of Ang II and TGF-&bgr;1. Therefore, in this study, we investigated the molecular mechanisms of how HGF antagonizes the Ang II-TGF-&bgr; axis in renal cells. In cultured human mesangial cells, TGF-&bgr;1 increased angiotensin type 1 receptor (AT1R) mRNA, mainly dependent on the Akt/phosphatidylinositol 3-kinase signaling pathway. Furthermore, TGF-&bgr;1 decreased the expression and phosphatase activity of phosphatase and tensin homolog, deleted on chromosome 10 (PTEN), a negative regulator of the phosphatidylinositol 3-kinase/Akt pathway. These data revealed positive feedback of the Ang II-TGF-&bgr; pathway, because Ang II increased TGF-&bgr; expression. In contrast, HGF significantly attenuated the increase in AT1R gene expression, and inhibited the decrease in PTEN induced by TGF-&bgr;1. Of importance, a PTEN-specific inhibitor significantly attenuated the reduction in TGF-&bgr;1–induced AT1R expression by HGF. These data suggest that HGF attenuated TGF-&bgr;1–induced AT1R expression through the PTEN/Akt pathway. To investigate this hypothesis, we performed in vivo experiments in mice with increased circulating levels of HGF produced by transgenically expressing HGF under control of a cardiac-specific transgene (HGF-Tg). In HGF-Tg mice, renal injury and fibrosis were significantly decreased, associated with reduction in AT1R expression and increase in PTEN after Ang II infusion, as compared with control mice. Moreover, these renal protective effects were abrogated by a neutralizing antibody against HGF. Thus, the present study demonstrated that HGF counteracts the vicious cycle of Ang II-TGF-&bgr;1-AT1R, mediating the inhibition of PTEN.

Selective Blockade of Periostin Exon 17 Preserves Cardiac Performance in Acute Myocardial Infarction

We previously reported that overexpression of full-length periostin, Pn-1, resulted in ventricular dilation with enhanced interstitial collagen deposition in a rat model. However, other reports have documented that the short-form splice variants Pn-2 (lacking exon 17) and Pn-4 (lacking exons 17 and 21) promoted cardiac repair by angiogenesis and prevented cardiac rupture after acute myocardial infarction. The apparently differing findings from those reports prompted us to use a neutralizing antibody to selectively inhibit Pn-1 by blockade of exon 17 in a rat acute myocardial infarction model. Administration of Pn neutralizing antibody resulted in a significant decrease in the infarcted and fibrotic areas of the myocardium, which prevented ventricular wall thinning and dilatation. The inhibition of fibrosis by Pn neutralizing antibody was associated with a significant decrease in gene expression of fibrotic markers, including collagen I, collagen III, and transforming growth factor-&bgr;1. Importantly, the number of &agr;-smooth muscle actin–positive myofibroblasts was significantly reduced in the hearts of animals treated with Pn neutralizing antibody, whereas cardiomyocyte proliferation and angiogenesis were comparable in the IgG and neutralizing antibody groups. Moreover, the level of Pn-1 expression was significantly correlated with the severity of myocardial infarction. In addition, Pn-1, but not Pn-2 or Pn-4, inhibited fibroblast and myocyte attachment, which might account for the cell slippage observed during cardiac remodeling. Collectively, these results indicate that therapeutics that specifically inhibit Pn exon-17, via a neutralizing antibody or drug, without suppressing other perisotin variants might offer a new class of medication for the treatment of acute myocardial infarction patients.

Inhibition of Lp(a)-induced functional impairment of endothelial cells and endothelial progenitor cells by hepatocyte growth factor.

BACKGROUND Lipoprotein (a) (Lp(a)) is one of the risk factors for peripheral artery disease (PAD). Our previous report demonstrated that hepatocyte growth factor (HGF) gene therapy attenuated the impairment of collateral formation in Lp(a) transgenic mice. Since risk factors for atherosclerosis accelerate endothelial senescence and impair angiogenesis, we examined the role of Lp(a) in dysfunction and senescence of endothelial progenitor cells (EPC) and endothelial cells. METHODS In vitro and in vivo incorporation assays were performed using ex-vivo expanded DiI-labeled human EPC. Senescence of cultured endothelial cells, production of oxidative stress and angiogenesis function were evaluated by SA-β-galactosidase staining, dihydroethidium (DHE) staining and Matrigel assay, respectively. RESULTS EPC transplantation significantly stimulated recovery of ischemic limb perfusion, while EPC pre-treated with Lp(a) did not increase ischemic limb perfusion. Impairment of angiogenesis by EPC with Lp(a) was associated with a significant decrease in CD31-positive capillaries and DiI-labeled EPC. Importantly, Lp(a) significantly accelerated the onset of senescence and production of reactive oxygen species (ROS) in human aortic endothelial cells, accompanied by a significant increase in the protein expression of p53 and p21. On the other hand, HGF significantly attenuated EPC dysfunction, senescence, ROS production, and p53 and p21 expression induced by Lp(a). CONCLUSION Lp(a) might affect atherosclerosis via acceleration of senescence, ROS production, and functional impairment of the endothelial cell lineage. HGF might have inhibitory effects on these atherogenic actions of Lp(a).

Endovascular therapy for subclavian artery restenosis due to stent strut protrusion with high-resolution angioscopy and three-dimensional optical frequency domain imaging

A 47-year-old female patient was admitted with a complaint of severe chest pain on effort. She had a history of effort angina treated using coronary artery bypass with left internal thoracic arterial bypass to the left ascending coronary artery. She also had left subclavian and vertebral arterial stenoses, which were treated with balloon-expandable stents. Exercise stress myocardial perfusion imaging revealed anterior to apex left ventricular myocardial ischemia. Cardiac ischemia due to left subclavian stenosis was diagnosed. We treated the left subclavian arterial stenosis with endovascular therapy. We observed that the vertebral Palmaz stent protruded from the ostium and the jailed subclavian artery on high-resolution angioscopy (Zemporshe with a 0.48-megapixel equivalent resolution; Taisho Biomed Instruments, Osaka, Japan) and optical frequency domain imaging (OFDI). A guide wire was successfully crossed through the Palmaz stent strut, which was confirmed using three-dimensional OFDI. The stent strut was dilated using balloon angioplasty. New imaging technologies are promising tools for improving the efficacy and safety of craniocervical intervention. <Learning objective: New imaging technologies including high-resolution angioscopy and 3D-optical frequency domain imaging have become available for endovascular therapy of peripheral artery disease. They could be used to observe stent strut deformation with high imaging quality and demonstrated successful guidewire passage through the stent strut. High-resolution angioscopic system yields images with higher quality and larger angular field than any other previous angioscopy systems.>

Refractory Vascular Wall Healing after Paclitaxel-Coated Nitinol Stent Implantation in the Femoropopliteal Artery: A High-Resolution Angioscopic Assessment

It is unclear whether arterial healing occurs beyond 1 year following paclitaxel-coated stent implantation in peripheral artery disease. An 81-year-old woman with superficial femoral artery disease underwent endovascular therapy with a paclitaxel-coated stent. An angiography 21 months later revealed peri-stent contrast staining in the superficial femoral artery, and optical frequency domain imaging demonstrated incomplete stent apposition with significant positive vascular remodeling. High-resolution angioscopy detected positive vascular wall remodeling and in-stent yellow plaque more clearly than conventional angioscopy. Refractory superficial femoral arterial wall healing was apparent more than 20 months after paclitaxel-coated stent implantation.

917 ROLE OF SEROTONIN IN ANGIOGENESIS: INDUCTION OF ANGIOGENESIS BY SARPOGRELATE VIA ENDOTHELIAL 5-HT1B/Akt/eNOS PATHWAY IN DIABETIC MICE

Objective: Serotonin (5-HT) plays a crucial role in pathological conditions of peripheral artery disease (PAD) and diabetes mellitus (DM). In these conditions, the balance between the 5-HT2A receptor in smooth muscle cells and the 5-HT1B receptor in endothelial cells (EC) regulates vascular tonus. In the present study, we focused on the role of 5-HT in endothelial dysfunction using a selective 5-HT2A receptor blocker, sarpogrelate. Method: To examine the role of 5-HT in diabetic PAD, a hindlimb ischemia model was created and sarpogrelate (50 mg/kg/day) was administered to streptozotocin (200 mg/kg) induced severe diabetic mice. Results: (in vitro) Stimulating human endothelial cells with 5-HT (10 &mgr;M) upregulates eNOS expression and phosphorylation of eNOS, Akt and ERK1/2 and increases tubule-formation on Matrigel. In contrast, high glucose significantly inhibited tubule formation, eNOS expression and increased apoptotic cell number, while 5-HT significantly attenuated these actions of high glucose (P < 0.01). These actions of 5-HT were inhibited by LY294002 (PI3K inhibitor). (in vivo) The ischemic blood flow was significantly lower in DM mice than in normal mice, while sarpogrelate significantly attenuated the decrease in the blood flow ratio compared to control (P < 0.01). Consistently, the decrease in eNOS expression and Akt activity in DM mice was significantly attenuated by sarpogrelate (P < 0.01). Conclusion: Present study demonstrated that selective inhibition of 5-HT2A by sarpogrelate significantly restored ischemic limb blood perfusion in a severe diabetic mouse model through stimulation of the eNOS/Akt pathway via the endothelial 5-HT1B receptor.